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Transdermal administration can be considered as an interesting route to overcome the side-effects inherent to oral intake. Designing topical formulations with maximum drug efficiency requires the optimization of the permeation and the stability of the drug. The present study focuses on the physical stability of amorphous drugs within the formulation. Ibuprofen is commonly used in topical formulations and then was selected as a model drug. Additionally, its low Tg allows easy, unexpected recrystallization at room temperature with negative consequence on skin penetration. In this study, the physical stability of amorphous ibuprofen was investigated in two types of formulations: (i) in terpenes-based deep eutectic solvents (DES) and (ii) in arginine-based co-amorphous blends. The phase diagram of ibuprofen:L-menthol was mainly analyzed by low-frequency Raman spectroscopy, leading to the evidence of ibuprofen recrystallization in a wide range of ibuprofen concentration. By contrast, it was shown that amorphous ibuprofen is stabilized when dissolved in thymol:menthol DES. Forming co-amorphous arginine–ibuprofen blends by melting is another route for stabilizing amorphous ibuprofen, while recrystallization was detected in the same co-amorphous mixtures obtained by cryo-milling. The mechanism of stabilization is discussed from determining Tg and analyzing H-bonding interactions by Raman investigations in the C=O and O–H stretching regions. It was found that recrystallization of ibuprofen was inhibited by the inability to form dimers inherent to the preferential formation of heteromolecular H-bonding, regardless of the glass transition temperatures of the various mixtures. This result should be important for predicting ibuprofen stability within other types of topical formulations.

International audience; L-Leucine is an essential amino acid which has been focusing a lot of investigations on its phase transition sequence for more than fifty years. Combining Raman spectroscopy and X-ray diffraction experiments provides a new interpretation of the second order phase transition extending between 270 and 360 K as a displacive incommensurate-normal phase transition. A soft mode was clearly detected from low-frequency Raman investigations which exhibits the temperature dependence (A·(TC−T)1/2) typical of the temperature behavior of the amplitudon, an excitation specific to incommensurate phases. Simultaneously to the softening of the amplitudon, several very weakly intense X-ray reflections vanish upon heating at 360 K, and thereby are interpreted as satellite reflections. This incommensurability was described as resulting from the freezing of thermally activated hydrophobic side-chain rotations upon cooling in disordered orientations. Raman investigations were also performed on the isomeric amino acid L-norleucine previously identified as undergoing a normal-incommensurate phase transition around 200 K. Comparison of both studies suggests that the temperature behavior of thermally activated local motions generates lattice instabilities. Loss of periodicity can result from the freezing of rotations of molecular moieties in disordered orientations, or from the enhancement of anharmonicity of these rotations. This could be a general phenomenon in hydrophobic amino acids with direct consequences on their applications in the life science area.

International audience; Mesoporous silica (MPS) carriers are considered as a promising strategy to increase the solubility of poorly soluble drugs and to stabilize the amorphous drug delivery system. The development by the authors of a solvent-free method (milling-assisted loading, MAL) made it possible to manipulate the physical state of the drug within the pores. The present study focuses on the effects of the milling intensity and the pore architecture (chemical surface) on the physical state of the confined drug and its release profile. Ibuprofen (IBP) and SBA-15 were used as the model drug and the MPS carrier, respectively. It was found that decreasing the milling intensity promotes nanocrystallization of confined IBP. Scanning electron microscopy and low-frequency Raman spectroscopy investigations converged into a bimodal description of the size distribution of particles, by decreasing the milling intensity. The chemical modification of the pore surface with 3-aminopropyltriethoxisylane also significantly promoted nanocrystallization, regardless of the milling intensity. Combined analyses of drug release profiles obtained on composites prepared from unmodified and modified SBA-15 with various milling intensities showed that the particle size of composites has the greatest influence on the drug release profile. Tuning drug concentration, milling intensity, and chemical surface make it possible to easily customize drug delivery.

International audience; Raman spectroscopy investigations on L-methionine (L-Met) performed in large temperature range (170 K – 420 K) and in a wide spectral window (5 – 3600 cm-1) have revealed an extended disordering mechanism triggered by thermally activated motions of the terminal side-chain atoms, from 250 K up to 390 K. This very progressive disordering process is characterized by two thermodynamic features, the first corresponding to a broad endotherm (250 K → 310 K) marking the beginning of the process while the second, ending the disordering transformation is a sharp endothermic peak at 390 K. These thermodynamic events are correlated with the softening of lattice vibrations and the increase of the quasielastic scattering, considered as the signatures of displacive phase transitions. The amorphous like band-shape of the low-frequency Raman spectrum collected above 390 K, resulting from the strong anharmonicity of local motions, is contrasting with the detection of additional Bragg peaks above 390 K by X-ray diffraction, consistent with the Cp jump accompanying the endothermic peak. These observations suggest that L-Met is progressively dynamically disordered adopting additional configurations in the crystalline lattice, through rotations of CH3 and the side-chain flexibility not clearly detected by X-ray diffraction. These results should be crucial for considering the stability of dried proteins composed of methionine residues.

International audience; The capabilities of the recent MAL (Milling-Assisted Loading) method were analyzed for the geometric confinement of amino acids within mesoporous silica matrices. The present study has shown a high filling degree (45 wt%) of L-tryptophan into unmodified MCM-41 pores. It was shown that the low-frequency Raman spectroscopy is a suited technique to determine that molecular materials are actually confined into silica-based ordered mesoporous materials from the analysis of lattice mode region, and to probe the molecular conformation of confined materials and their interaction with the pore surface from the analysis of internal vibrations.

International audience; Using the innovative solid-state loading (milling-assisted loading, MAL) method to confine caffeine to cylindrical pores (SBA-15, B ¼ 6 nm) gives the opportunity to explore the original physical states of caffeine and their subsequent transformation using low-frequency Raman spectroscopy, powder X-ray diffraction and microcalorimetry investigations. It was shown that MAL makes possible the loading of the selected form in the polymorphism of caffeine. While form II has similar structural and dynamics properties in confined and bulk forms, the confined rotator phase (form I) exhibits clear differences with the bulk form inherent to its orientational disorder. Interestingly, the two confined forms of caffeine undergo an exothermic disordering transformation upon heating into a physical state at the border between a nanocrystallized orientationally disordered phase and an amorphous state, not existing in the bulk form. The melting of this new physical state was observed at 150 C, i.e. 85 degrees below the melting temperature of the bulk form I, thus demonstrating the confinement of caffeine. It was alsofound that the liquid confined to pores of 6 nm mean diameter recrystallizes upon cooling, which can be explained by the very disordered nature of the recrystallized state.

Mesoporous silica carriers have recently gained interest in the pharmaceutical domain because of their potential to significantly increase the solubility of poorly water-soluble drugs, by adsorbing the active molecule in an amorphous and relatively stable state. However, the use of organic solvents for drug loading and the limited loading capacity are real drawbacks in the context of therapeutic use. The present work shows that co-milling of porous SBA-15 matrix with the drug makes it possible the drug loading without significant damage for the structure of the matrix, enhancing thereby the capacity of loading to almost 40 wt%. Combining calorimetric, thermogravimetric, and low-frequency Raman investigations has allowed us to determine optimum conditions of loading, and to analyze the physical state, the molecular mobility and the phase transformations of the confined drug upon heating. It was shown that the physical state of ibuprofen (IBP) confined to silica carriers was amorphous between temperatures T g and T m of the bulk form of IBP.

International audience; The nonionic Type V deep eutectic solvent (DESs) thymol + menthol is experimentally and computationally studied aiming to clarify the relation between its liquid phase structure andits thermodynamic nonideality. 1H NMR, Raman, and X-ray scattering analysis of the thymol + menthol system, supported by molecular dynamics simulations, show complex intermolecularinteractions dominated by sterically hindered H-bonded clusters. For temperatures greater than or equal to room temperature, a quasi-linear evolution of the eutectic system properties between the pure compounds is observed, suggesting the absence of a magic stoichiometric composition in the eutectic solvent. However, temperature dependent Raman spectroscopy indicates a notable increase in thymol−menthol H-bonding as temperatures approach the eutectic point. This study shows that nonionic Type V DESs present an important temperature-dependent nonideality originating from the change in the intermolecular H-bonding withtemperature. These findings have significant implications for the design and growing application of Type V DESs.

The phase diagram of the xylitol – citric acid system was determined from microcalorimetry experiments performed on mixtures prepared by co-milling. Raman investigations in the stretching regions of carbonyl and hydroxyl groups have shown a close relationship between the formation of molecular associations between the two molecular species and the drastic negative deviation from ideality for high citric acid content, responsible for obtaining a deep eutectic solvent (DES). It was shown that a great amount of poorly water-soluble drugs (ibuprofen and paracetamol) could be dissolved in that DES, providing a strategy for stabilizing a great amount of amorphous drugs or for obtaining unusual crystalline forms of drugs.

International audience; In this paper, we present a kinetic investigation of the polymorphic transformation γ → α of sorbitol under milling in the objective to identify the microscopic mechanisms that govern this type of solid-state transformation. The milling was performed with a high energy planetary mill and the milled material was analysed by DSC, PXRD and Raman spectrometry. The transformation kinetics was found to be sigmoidal with a noticeable incubation time. Moreover, this incubation time was shown to shorten rapidly when seeding the initial form γ with the final form α. The origin of the incubation period and its evolution upon seeding are puzzling as polymorphic transformations induced by milling are not expected to occur through a nucleation and growth process. To explain these puzzling kinetic features, we propose a two-step transformation mechanism involving local amorphisations due to the mechanical impacts, immediately followed by rapid recrystallizations of the amorphized fractions. The key point of the mechanism is that recrystallizations are oriented towards the forms γ or α, depending on the crystalline form of neighbouring crystallites. This mechanism has been validated by numerical simulations which were able to reproduce all the experimental kinetic features of the polymorphic transformation (kinetic law and effects of seeding) upon milling.

International audience; Using the Milling-Assisted Loading (MAL) solid-state method for loading a poorly water-soluble drug (ibuprofen, IBP) within the SBA-15 matrix has given the opportunity to manipulate the physical state of drugs for optimizing bioavailability. The MAL method makes it easy to control and analyze the influence of the degree of loading on the physical state of IBP inside the SBA-15 matrix with an average pore diameter of 9.4 nm. It was found that the density of IBP molecules in an average pore size has a direct influence on both the glass transition and the mechanism of crystallization. Detailed analyzes of the crystallite distribution and melting by Raman mapping, x-ray diffraction, and differential scanning calorimetry have shown that the crystals are localized in the core of the channel and surrounded by a liquid monolayer. The results of these complementary investigations have been used for determining the relevant parameters (related to the SBA-15 matrix and to the IBP molecule) and the nature of the physical state of the confined matter.

International audience; A new method for determining solubility lines of drugs in polymers, based on low-frequency Raman spectroscopy measurements, is described and the results obtained by this method are compared with those obtained using a more classical method based on differential scanning calorimetry investigations. This method was applied to the paracetamol/PVP system using molecular and crystalline dispersions (MCD) rather than usual physical mixtures to reach faster the equilibrium saturated states and make the determination of the solubility line more rapid.

The bioprotective properties of 2 disaccharides (sucrose and trehalose) were analyzed during the freeze-drying (FD) process and at the end of the process, to better understand the stabilization mechanisms of proteins in the solid state. In situ Raman investigations, performed during the FD process, have revealed that sucrose was more efficient than trehalose for preserving the secondary structure of lysozyme during FD, especially during the primary drying stage. The lower bioprotective effect of trehalose was interpreted as a consequence of a stronger affinity of this disaccharide to water, responsible for a severe phase separation phenomenon during the freezing stage. Dielectric spectroscopy investigations on the freeze-dried state of protein formulations have shown the capabilities of trehalose assisted by residual water to reduce the molecular mobility of the vitreous matrix, suggesting that trehalose is more efficient to preserve the protein structure during long-term storage.

International audience; A very unusual amorphous-amorphous two-step transformation was observed in indomethacin confined within MCM-41 mesoporous silica, via isothermal and non-isothermal routes. Isothermally, at room temperature, the first step corresponds to the 1-dimensional growth of gamma-nanocrystals along the mesoporous channels from preexisting incipient nuclei. In the second step nanocrystals slowly transform into an undercooled liquid state structurally different from the original liquid and characterized by a higher molecular mobility. The instability of nanocrystals detected in the continuation of the isothermal growth was explained as resulting from a very high specific area of nanocrystals imposed by the geometrical confinement when the size of nanocrystals along the channels achieves a dimension much larger than the channel diameter. The mechanism of the slow dissolution of nanocrystals was identified as similar to a digestive ripening process, inverse to the Ostwald ripening, by which larger clusters dissolve to the detriment of smaller ones. This two-step transformation was also observed upon heating. In this case, the growth of nanocrystals is followed by a melting far below the melting point of gamma-crystals, according to Gibbs-Thomson effects. This study shows that the original amorphous form of indomethacin transforms into a second amorphous form via the transient growth of nanocrystals which become unstable via two different mechanisms induced by the geometrical confinement.

Micro-Raman spectroscopy gives the original opportunity to monitor simultaneously the operating process and the protein structure from in situ investigations along the 3 stages of the freeze-drying (FD) process. This opportunity was used for determining how a small amount of glycerol enhances the bioprotective efficiency of trehalose during FD of lysozyme formulations. Three lysozyme formulations were analyzed: lysozyme dissolved in D2O (wt% 1:9), in trehalose-D2O mixture (wt% 1:1:8), and in the trehalose-glycerol-D2O mixture (wt% 1:1:0.17:7.93). Raman mapping performed during each stage of the FD process has provided important information about the preferential interaction between water, trehalose, and lysozyme in relation to the protein stability. It was found that the addition of a small amount of glycerol had a plasticizing effect on the glassy trehalose-water matrix during the primary drying stage and then reduced the bioprotective effect of trehalose. By contrast, during the secondary drying stage, glycerol significantly enhanced the stabilizing effect of trehalose in the same sample, by replacing water-trehalose H-bonds with stronger glycerol-trehalose H-bonds and stiffening the amorphous trehalose matrix. The action of glycerol is also related to its capability to prevent aggregation of trehalose, making the structure of the frozen product more homogeneous, by changing the hydrogen-bond networks in the liquid formulation before the freezing stage.

In this work, three monomeric forms of arabinitol, usually named arabitol, and their dimeric species have been structural and vibrationally studied by using the micro-Raman spectra in the solid phase accomplished with theoretical calculations based on the theory of the functional of the density (DFT). The hybrid B3LYP method was used for all the calculations together with the 6-31G* and 6-311++g** basis sets. Two different L structures with minima energies were predicted in accordance to the two polymorphic structures revealed by recent X-ray diffraction experiments. The studies by natural bond orbital (NBO) calculations reveals high stabilities of the L form as compared with the D one but the topological properties by using the atoms in molecules (AIM) suggest a higher stability of the D form due to a strong H bond interactions. The scaled mechanical force fields (SQMFF) procedure was used to perform the complete vibrational assignments for the monomeric forms and their dimer. On the other hand, the similarity in the gap values computed for the three forms of arabitol with those observed for sucrose, trehalose, maltose and lactose in gas phase at the same level of theory could partially explain the sweetening property of this alcohol. In addition, the influences of the size of the basis set on some properties were evidenced.

Valsartan is an antihypertensive drug, recognized to be marketed in an amorphous state, different from that obtained by quenching the liquid state below Tg. This is an unusual and very original situation, given that the amorphous state is unstable. Low-wavenumber Raman spectroscopy and X-ray diffraction investigations were carried out on the various solid-state forms of valsartan. It was found that the marketed form is not amorphous and can be described in terms of mesophase in which the long-range order of the stable crystal is limited on the lengthscale of tens nanometers, inducing a melting temperature lower than that of the stable crystalline state, inherent to the crystallite size. This unusual physical state of a marketed drug was correlated to the relative population of cis-trans conformers, preventing the development of the hydrogen-bond network distinctive of the long-range order in the crystalline state.

The present study focuses on protein motions on the picosecond time scale, generally characterized by the overlapping of vibrational and relaxational dynamics in disordered molecular systems. Recently, it has been demonstrated that a dry protein, bovine serum albumin (BSA), shows a glass-like transition in the temperature range between 240 and 260 K. Here, we present the results of combined low-frequency Raman and inelastic neutron scattering studies of dry BSA under conditions similar to those of this glass-like transition. The use of both techniques allows us to perform a detailed comparison of the dynamic susceptibility and vibrational density of states of BSA obtained at different temperatures and to calculate the light-vibration coupling coefficient, C(ω). Moreover, we analyzed the temperature evolution of the boson peak and a peak located at ∼80 cm–1, which has previously been identified to originate from protein dynamics, and observed that both modes show anomalous temperature behavior in the vicin...

International audience; The polymorphism of sulindac was investigated by Raman investigations, mainly in the low-wavenumber region in order to analyze the influence of the amorphization method on recrystallization and crystalline form stability. By devitrification of the quenched liquid, it was found that the undercooled liquid crystallizes into Form I, and a polymorphic transformation by cooling Form I toward Form IV, was clearly revealed. The low-wavenumber spectra of polymorphic forms are direct fingerprints of crystals, indicating a degree of disorder of Form IV intermediate between those of the ordered Form II (commercial form) and the relatively disordered Form I. This study has shown the enantiotropic relationship between Forms I and IV and that both the temperature of crystallization and the physical stability of Form I prepared is dependent on the technique used for preparing amorphous sulindac.

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Kinetics of isothermal crystallization of l-arabitol were analyzed from the undercooled liquid state within micron-sized droplets from micro-Raman spectroscopy. This study reveals that crystallization slightly above Tg is controlled by stochastic heterogeneous nucleation inherent to the droplet size. Microscopic Raman investigations performed in droplets give the unique opportunity to analyze the pure metastable Form II of l-arabitol. It was found that Form II is characterized by a molecular packing more compact than that of the stable Form I, inherent to strong intermolecular hydrogen bonding. Kinetics laws obtained by analyzing several droplets at different temperatures, reveal the transient character of Form II, quasi systematically detected during the crystallization process of form I. Form II appears as the first step of crystallization prior to successive short-living metastable states which is necessary to achieve a complete crystallization in Form I. It was found that the kinetics of conversion between the metastable states (Form II) into Form I is dependent on the amount of strong hydrogen bonding distinctive of Form II.

The physical state of amorphous powder obtained by cryomilling forms I and III of carbamazepine (CBZ) were analyzed from low-wavenumber Raman spectroscopy investigations and compared to that of the quenched liquid. This analysis has shown subtle structural modifications between the amorphous states prepared by melt-quenching and cryomilling polymorphs I and III of CBZ. Moreover, two different non-isothermal crystallization mechanisms from these two different types of amorphous states were revealed, in agreement with calorimetric analyzes. Raman spectroscopy and differential scanning calorimetry experiments performed on cryomilled forms I and III of CBZ, provide information on the bimodal crystallization, observed upon heating several amorphous organic crystalline materials produced by milling. This study clearly shows that milling gives the opportunity to explore new amorphous states which cannot be achieved using the classical melt-quenching method.

The coating of a nonwoven textile by polyelectrolyte multilayer film (PEM) issued from cationic and anionic β-cyclodextrin (βCD) polyelectrolytes according to the layer-by-layer (LbL) technique was successfully attempted. The tert-butyl benzoic acid (TBBA) was used as drug model to evaluate the loading capacity and sustained release properties of this PEM system. The build-up of the multilayer assembly was monitored in situ by optical waveguide lightmode spectroscopy (OWLS) on the one hand, and was assessed by gravimetry on the other hand when applied onto the textile substrate. In parallel, the complexation study of TBBA with both CD polyelectrolytes was also investigated by nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC). The influence of thermal crosslinking of the multilayered coating on its stability and on TBBA release kinetics in phosphate buffered saline (PBS) at 37°C was studied. Finally, biological and microbiological tests were performed to investigate the cytocompatibility and the intrisic antibacterial activity of multilayer assemblies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1408-1424, 2016.

The influence of HPβCD on the thermal denaturation of lysozyme was analyzed mainly from microcalorimetry and Raman investigations carried out in the molecular fingerprint and the low-frequency regions. It was shown that Raman spectroscopy investigations performed on a wide spectral range give the opportunity to describe the influence of HPβCD on the mechanism of protein denaturation. Using D2O as solvent allowed us to show that HPβCD mainly destabilizes the tertiary structure of lysozyme by enhancing the protein flexibility and thus inducing the destabilization of the secondary structure. Principal components analysis (PCA) was used for spectra treatment, providing important information about inclusion complex formation between protein hydrophobic residues and CDs molecules. Combining PCA and classical technics (curve fitting) of data analysis allowed a better understanding of the influence of HPβCD on the protein denaturation that seems to be related to the CDs capacity to form inclusion complex. It was observed that these interactions prevent the formation of new strong H-bonds between β-sheet structures thereby inhibiting protein aggregation. This study reveals that CDs are promising systems for inhibiting protein aggregation without protein denaturation, only if designing derivative CDs is carefully controlled.

International audience; Metoprolol tartrate and metoprolol free base loaded pellet starter cores were coated with Eudragit RS, plasticized with 25% triethyl citrate (TEC). The initial drug loading and coating level were varied from 10 to 40 and 0 to 20%, respectively. Drug release was measured in 0.1 N HCl and phosphate buffer pH 7.4. The water uptake and swelling kinetics, mechanical properties and TEC leaching of/from coated pellets and/or thin, free films of identical composition as the film coatings were monitored. The following unusual tendencies were observed: (i) the relative drug release rate from coated pellets increased with increasing initial drug content, and (ii) drug release from pellets was much faster for metoprolol free base compared to metoprolol tartrate, despite its much lower solubility (factor >70). These phenomena could be explained by plasticizing effects of the drug for the polymeric film coatings. In particular: 1) Metoprolol free base is a much more potent plasticizer for Eudragit RS than the tartrate, leading to higher film permeability and overcompensating the pronounced differences in drug solubility. Also, Raman imaging revealed that substantial amounts of the free base migrated into the film coatings, whereas this was not the case for the tartrate. 2) The plasticizing effects of the drug for the film coating overcompensated potential increasing limited solubility effects when increasing the initial drug loading from 10 to 40%. In summary, this study clearly demonstrates how important the plasticization of polymeric controlled release film coatings by drugs can be, leading to unexpected formulation effects.

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. In this work, we provide new insight on the influence of the precursor (CH 4 vs. C 2 H 2 ) on the mechanical properties of a-C:H coatings synthesized in a hybrid PECVD/PVD process. For a given set of plasma parameters, the films synthesized in CH 4 /Ar gas mixture are found harder than the ones prepared in C 2 H 2 /Ar atmosphere despite their lower sp3 density and similar hydrogen content. This unusual behavior is attributed to different molecular organization of the polymeric network constituting the films. The latter is explained considering the influence of the chemical nature of the film-forming species on the cross-linking and branching degree of the films. The whole set of our results unambiguously demonstrates that additional factors than the sp3 and hydrogen content have to be considered for explaining the mechanical properties of a-C:H layers.

We describe a method for analyzing protein hydration by Raman spectroscopy on the model protein lysozyme. The analysis of the protein hydration shell is made possible by dissolving the protein in D2O, providing via isotopic exchange the uncoupled O – H stretching spectrum of water molecules early bound to the protein, which are thereafter spread into the solvent. The spectrum of the hydration water can be obtained by subtracting the spectrum of the contribution of D2O from that of the aqueous lysozyme solution in the intramolecular O – D stretching vibrations region (2,200–2,800 cm−1). Raman investigations were simultaneously carried out in the amide I region (1,500–1,800 cm−1) and in the O – D/H stretching spectrum (3,200–3,800 cm−1) during thermal denaturation of lysozyme, to analyze structural changes of the protein in relation to the physical properties of hydration water. It was found that the H-bond network of hydration water is slightly distorted compared to the bulk water at room temperature, with a loss of the tetrahedral local order. The difference between hydration and bulk water is significantly enhanced at T = 90 °C in the denaturated state of the protein. The quantification of water molecules in direct interaction with the protein provides the temperature dependence of the solvent-accessible surface area during the denaturation process. Both kinds of information on hydration water and protein structure lead to a detailed description and overall understanding of the mechanism of protein denaturation.

The crystallization process of l -arabitol was investigated using differential scanning calorimetry (DSC) and X-ray diffraction. In order to estimate the temperature range where the rates of nucleation and growth reach their maximum, we have applied an experimental protocol consisting in a succession of quenching of the liquid at different temperatures followed by a heating ramp. It enabled us to show that the nucleation and growth domains were clearly separated and a metastable polymorph of lower melting point was obtained. The conditions for the appearance of this form have been studied in detail. The crystallization kinetic of the undercooled liquid at T = 0 °C, followed by X-ray diffraction, has permitted to show that the stable form was obtained after the transient metastable form.

Background Recently, it has been revealed that tetragonal lysozyme crystals show a phase transition at 307 K upon heating. The underlying mechanisms of the phase transition are still not fully understood. Here we focus on the study of high-frequency vibrational modes arising from the protein and their temperature evolution in the vicinity of Tph as well as on the detailed study of crystalline water dynamics near Tph. Methods Raman experiments have been performed at temperatures 295–323 K including Tph. The low-frequency modes and the modes of fingerprint region, CH- and OH-stretching regions have been analyzed. Results and conclusions In spite of the absence of noticeable rearrangements in protein structure, the high-frequency vibrational modes of lysozyme located in the fingerprint region have been found to exhibit the features of critical dynamics near Tph. Pronounced changes in the dynamics of α-helixes and Tyr residues exposed on the protein surface point to the important role of H-bond rearrangements at the phase transition. Additionally the study of temperature evolution of OH-stretching modes has shown an increase in distortions of tertahedral H-bond network of crystalline water above Tph. These changes in water dynamics could play a crucial role in the mechanisms of the phase transition. General significance The present results shed light on the mechanisms of the phase transition in lysozyme crystals.

Raman investigations were carried out in situ in real time during the lyophilization of three proteins (β-lactoglobulin, bovine serum albumin, and chymotrypsinogen) characterized by different structural properties. Structural changes in the proteins were only and systematically detected after the primary drying step of the lyophilization, through a frequency shift and a general broadening of amide I and III bands. These spectral changes have been interpreted in terms of local disordering related to the distortion of the structural elements induced by ice desorption. Structural changes of the secondary structure were found almost reversible upon rehydration, whereas changes in the solvent accessibility to protein residues are detected and related to the alteration of the tertiary and/or quaternary structures. The influence of the lyophilization parameters, corresponding to different stress conditions, on the degree of protein denaturation has been analyzed.

Low- and high-frequency Raman experiments in the 5-200 cm(-1) and 600-1800 cm(-1) ranges were carried out in the crystalline and amorphous states of ibuprofen. Low-frequency investigations indubitably reveal the existence of a molecular disorder in the metastable phase (phase II), through the observation of quasielastic contribution below 30 cm(-1), and the absence of phonon peaks in the Raman susceptibility which mimics the density of vibrational states of an amorphous state. High-frequency Raman spectra indicate a local order in phase II similar to that in the glassy state. Both dynamic and static molecular disorder could contribute to the Raman signatures of the disorder in crystalline phase II. Raman investigations suggest that phase II can be considered as a transient metastable state in the devitrification process of ibuprofen upon heating from a far from equilibrium state toward the stable phase I.

We show in this paper the contribution of the whole Raman spectrum including the phonon spectrum, to detect, identify and characterize polymorphic forms of molecular compounds, and study their stability and transformation. Obtaining these kinds of information is important in the area of pharmaceutical compounds. Two different polymorphic systems are analyzed through investigations in indomethacin and caffeine exposed to variable environmental conditions and various stresses, as possibly throughout the production cycle of the active pharmaceutical ingredient. It is shown the capability of the low-frequency Raman spectroscopy to reveal disorder in the crystalline state, to detect small amorphous or crystalline material, and to elucidate ambiguous polymorphic or polyamorphic situations.

Cold- and heat-induced β-lactoglobulin (BLG) transformations have been analyzed in the presence of 4 M urea, from Raman spectroscopy investigations carried out simultaneously in the low wavenumber range (10–400 cm−1) and in the amide I region (1500–1800 cm−1). These investigations show common features between the denaturation processes at low and high temperatures. The denatured states are reached via an intermediate state characterized by a soft tertiary structure without detectable conformational changes. This intermediate is intimately connected with a tetrahedral hydrogen-bond structure of water which extends over a limited range. It is shown that the disruption of the hydrogen-bond network of D2O has an important consequence on the solvent dynamics, which controls protein dynamics and is characterized by an anharmonic behavior. By monitoring the amide I mode, conformational changes are detected at low temperature (below 5 °C) and determined to be similar to those detected at high temperature in the presence of urea near 65 °C, and in the absence of urea near 80 °C. The conformational changes are described as a loss of α-helix structures and a concomitant formation of β-sheets. The temperature dependence of the amide I wavenumber in BLG dissolved in the 4 M urea aqueous solution was interpreted on the basis of a two-state model, leading to the protein stability curve related to its molecular conformation. Copyright © 2011 John Wiley & Sons, Ltd.

It has been recently shown that mechanical milling can amorphize D-glucose without any mutarotation, giving rise to an anomerically pure amorphous sample. We have taken advantage of this exceptional possibility to study the kinetic of mutarotation in the amorphous solid state. The investigations have been performed in situ by time-resolved Raman spectroscopy. The results reveal an unexpected coupling between the mutarotation process and the structural relaxations involved in the glassy state.

The structural and thermodynamic changes in the organic molecular crystals of α -D-glucose and β -D-glucose upon high energy ball milling at room temperature and −15 ∘C have been studied. The investigations have been performed by X-ray diffraction, differential scanning calorimetry and Raman spectroscopy. The results show the possibility to reach the glassy amorphous state of both forms of glucose, if mechanical milling is performed far enough below the glass transition temperature of the corresponding liquid ( T g ≈ 38 ∘ C ) . This route to the glassy state is found to be free of mutarotation whatever the starting glucose variety, while this mutarotation is unavoidable using the usual thermal quench of the liquid.

The coating of a nonwoven textile by polyelectrolyte multilayer film (PEM) issued from cationic and anionic β-cyclodextrin (βCD) polyelectrolytes according to the layer-by-layer (LbL) technique was successfully attempted. The tert-butyl benzoic acid (TBBA) was used as drug model to evaluate the loading capacity and sustained release properties of this PEM system. The build-up of the multilayer assembly was monitored in situ by optical waveguide lightmode spectroscopy (OWLS) on the one hand, and was assessed by gravimetry on the other hand when applied onto the textile substrate. In parallel, the complexation study of TBBA with both CD polyelectrolytes was also investigated by nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC). The influence of thermal crosslinking of the multilayered coating on its stability and on TBBA release kinetics in phosphate buffered saline (PBS) at 37°C was studied. Finally, biological and microbiological tests were performed to investigate the cytocompatibility and the intrisic antibacterial activity of multilayer assemblies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1408-1424, 2016.

This study shows the contribution of Raman spectroscopy to carefully describe the mechanism of solid-state transformation at the molecular level, by simultaneously monitoring the molecular reorganization and the water escape during the dehydration kinetics. Dehydration of monohydrate caffeine was analyzed at various temperatures. In-situ low- and high-frequency Raman investigations provide information on the long-range and local arrangement of caffeine molecules, during the dehydration kinetics and in the anhydrate state. The nature of the anhydrate was determined, from the analysis in the low-frequency region, as metastable and transient between both polymorphic forms of anhydrous caffeine. It was shown that changing the temperature mainly induces structural reorganization of caffeine molecules in the hydrate state, leading to an anhydrate more or less transformed in form I of anhydrous caffeine.

A detailed structural description of the glacial state in triphenyl phosphite, based on Raman microspectroscopy investigations and a complementary structural analysis by X-ray diffraction, of a nano-structured glacial state, is reported in this paper. The optical images obtained for the glacial state, isothermally prepared at 214 K and 220 K, display contrasting effects typical of an heterogeneous system. Very large clusters of hexagonal shape are distinguished, and Raman microspectroscopy gives the opportunity to describe the structural organization inside these clusters. The diffraction pattern of a glacial state formed in a low-temperature range [210 K, 216 K], was calculated from the consideration of an isotropic crystallite size of 50 A. The good agreement between the calculated and experimental diffraction patterns confirms that the glacial state is composed of crystallites of the stable crystalline phase coexisting with the non-transformed supercooled liquid according to the micro-structural analysis of a glacial state prepared at higher temperature (222 K). Raman spectroscopy and X-ray diffraction investigations provide the description of two structural organizations corresponding to the liquid and the crystalline states. No other structural organization was detected in TPP using (direct and indirect) structural probes.

Solid deposits have been formed at 88 K and 10−1 Torr from ethanol–water gas collected above aqueous solutions of ethanol (EtOH) (0.6, 2, 4.5, 9 and 17 mol%). The composition of different gas mixtures varying between 1:16 and 1:0.8 EtOH:H2O are determined at 295 K using our experimental vapor–liquid equilibrium (VLE) data in combination with the Wilson model [28] . The Wilson constants derived at this temperature are Λ12 = 0.37(4) and Λ21 = 0.58(5). The concentration of EtOH in the ice mixture can be calculated using these data and a kinetic model of condensation. It is found to vary between 9 and 65 mol% EtOH. The ice mixtures are analyzed in situ in a modified cryostage by micro-Raman spectroscopy. The distinct vibrational signatures of pure EtOH, EtOH aqueous solutions and EtOH–ice mixtures are identified in the 400–3800 cm−1 spectral range. Internal vibrational motions of EtOH molecules are affected by temperature and concentration. The presence of amorphous EtOH–ice phases at 88 K is demonstrated by the characteristic vibrational signatures of the νOH stretching modes. The crystallization of an EtOH hydrate is proposed during annealing at ∼140 K of a 65 mol% EtOH–ice mixture. According to our preliminary X-ray diffraction work, this phase has apparently a distinct structure from that of solid EtOH or from EtOH–clathtrate structures usually found in frozen aqueous solutions. For ice mixtures of lower EtOH content, a distinct hydrate phase crystallizes at ∼170 K. These results suggest that ice mixtures obtained by vapor deposition reflect the existence of EtOH clusters of a distinctive structural nature with respect to those encountered in frozen aqueous mixtures.

Kinetic investigations of the polymorphic form alpha of anhydrous trehalose have been performed below its apparent melting temperature (Tm) by differential scanning calorimetry (DSC) and X-ray diffraction. The results reveal a spontaneous isothermal vitrification process which indicates that the phase alpha is in a very unusual superheating situation. This behavior has been attributed to the fact that the effective melting temperature (Tm(eff)) of the phase alpha is likely to be located far below the glass transition temperature (Tg) of this compound. The high viscosity of the liquid trehalose between Tm(eff) and Tg is thus invoked to explain the long lifetime of the phase alpha in this temperature range.

The structure and microstructure (refinement of the isotropic size and microstrain parameters) of the glacial state in triphenyl phosphite (TPP, P(OC6H5)3) transformed at 222K have been determined from powder synchrotron X-ray diffraction data through a Rietveld and a Le Bail refinement, respectively. It is shown that the glacial state is composed of crystallites of the stable crystal phase coexisting with non-transformed supercooled liquid, the apparent size of the crystallites—depending on the aging temperature at which the glacial state is isothermally formed, [Phys. Rev. B 60 (1999) 9390]—being equal to 329.2(2) A at 222K. The molecular conformation is slightly less mirror-symmetric than the one in the crystal state, and correlatively only one of the two unusual weak intermolecular C–H⋯O hydrogen bonds already observed in the latter state is encountered in the glacial one. Additional Raman scattering investigations confirm the previous result and reveal in addition that no hydrogen bonding interaction is observed neither in the glass nor in the liquid states.

Raman scattering investigations of cyanoadamantane (CNa) plastic crystals were performed in the 0?10?GPa pressure range. A phase transition towards an ordered high-pressure (HP) phase is clearly identified. However, this transition can be easily bypassed upon pressurization, placing the initial plastic phase in a metastable situation. Between 0.3 and 0.7?GPa the escape from metastability is observed through the isobaric transformation towards the ordered HP phase. Above 0.7?GPa, the lifetime of the metastability appears to be infinite with respect to the experimental timescale, suggesting that the glassy crystal state was reached upon pressurization. Above 10?GPa, the metastable plastic phase, as well as the ordered HP phase, convert into a conventional amorphous state. The structural features of the ordered and the glassy crystal HP phases, determined from Raman investigations, were compared to those of the ordered and glassy crystal phases usually obtained upon cooling.

Raman scattering investigations of cyanoadamantane plastic crystal in a narrow pressure range (0–1 GPa), reveal the evidence for a kinetic transformation of the disordered state under pressure towards an ordered high-pressure (H-P) phase. This latter is observed to be different from the ordered low-temperature phase. The ability of cyanoadamantane to be placed in a disordered metastable state on pressurization suggests the existence of a glass transition under pressure. The order–disorder phase transition was observed on the decreasing pressure range of 0.3–0.2 GPa. The close relation between the low-frequency Raman spectrum of the orientational glass obtain by rapid cooling of the plastic phase and the lattice modes of the ordered H-P phase could indicate that this latter is involved in the vitrification process of cyanoadamantane.

We report Raman investigations on stable and metastable phases of cyanoadamantane plastic crystal, which gives an orientational glassy state upon a deep undercooling below 175 K. The isothermal transformation of the metastable quenched plastic phase is systematically analyzed for quenches in the 180-250 K temperature range. Two different kinds of ordering process have been evidenced, depending on the depth of the quench. For shallow quenches (above 200 K) the ordering process is characterized by sigmoidal growth curves corresponding to the nucleation and growth of the ultimately stable state. For deep quenches (below 200 K), atypical kinetics laws are observed. In this case the system escapes from its metstable disordered state preferentially via the transient development of domains whose structure slightly differs from that observed in the ultimately stable phase, in agreement with Ostwald's rule of stages. The development of this local structure, after quenches at temperatures approaching Tg, is considered to be responsible for the extension of the lifetime of the metastability of the plastic phase and then could be at the origin of the glass formation.